Influence of RORγt expression in Tregs for the inhibition of colitis onset

Our previous data have unraveled an increased suppressive capacity of RORγt⁺ Foxp3⁺ T cells in comparison to RORγt^- Foxp3⁺ T cells in experimental colitis. For this reason we addressed the question whether the increased suppressive capacity of RORγt⁺ Foxp3⁺ T cells is RORγt dependent. We could demonstrate that RORγt⁺ Foxp3⁺ T cells could be induced from RORγt^- Foxp3⁺ T cells; however, the influence of RORγt expression on the observed changes in the gene expression profile and functional capacities in comparison to Foxp3⁺ T cells remains unclear. A previous study has demonstrated the critical influence of the expression of the transcription factor Stat3 for the suppressive capacity of Tregs in Th17 associated inflammatory responses¹⁰⁹. Stat3 expression is induced by IL-6, independent from RORγt expression²³⁴. However, the expression of RORγt could influence the chemokine receptor repertoire as CCR6 expression could be promoted by the RORγt expression²³⁵.

In order to investigate the effect of RORγt expression of Tregs on their suppressive capacity in colitis settings, we performed a transfer colitis experiment, sorting and transferring naïve T cells from CD90.1 C57BL/6 mice into RAG2 KO recipients. Additionally, CD90.2⁺ CD4⁺ CD25^high T cells were isolated from RORc^WT/WT or RORc^KO/KO mice and co-transferred in a ratio of 1:4 (Treg : Tn). Previous studies have demonstrated a reduction of αβ T cells subsets in the intestinal tissue in RORc^KO/KO mice in comparison to wildtype mice; however, no influence on the functional capacities and the survival of peripheral Tregs in these mice have been described until now⁸⁵. We could not observe any changes in either frequencies or numbers of total Foxp3⁺ T cells in RORc^KO/KO mice in the peripheral lymphoid organs (data not shown). Therefore, CD4⁺ CD25^high T cells isolated from these mice were assumed to represent a functional Treg population. This hypothesis could be confirmed by the observed reduction of colitis onset in mice transferred with either RORc^WT/WT or RORc^KO/KO Tregs (Figure 3.17 A). Additionally, we detected a reduced histopathological score in the colon of mice co-transferred with RORc^WT/WT or RORc^KO/KO Tregs in comparison to the control group (Figure 3.17 B) as well as a reduced total cell number in the colon (Figure 3.17 C). We could not detect any differences in the influence of RORc^WT/WT and RORc^KO/KO Tregs on disease severity, as both led to a reduced colitis pathology. This could be due to the low frequency (~

1-2 %) of RORγt⁺ Foxp3⁺ T cells in the population of total Foxp3⁺ Tregs isolated from a pool of spleen and lymph node cells. In order to analyze whether we could observe changes in the migratory or functional capacity of RORc^WT/WT and RORc^KO/KO Tregs we determined their frequency and number in the analyzed organs and their influence on the population of Teff

cells (Figure 3.18). We could not observe any significant differences in the frequency and number of CD90.2⁺ T cells between the two groups in the colon or the other analyzed organs

0 1 2 3 3 4 5 6 7

% of initial body weight PBS TregsWT/WT + RORc TregsKO/KO + RORc

T_n

Figure 3.17: RORγt-deficient Tregs exhibit a similar suppressive capacity in transfer colitis in comparison to WT Tregs.

Sorted CD4⁺ CD45RB^high naïve T cells from CD90.1 C57BL/6 mice were injected intraperitoneally into RAG2 KO mice to induce colitis. Additionally, CD4⁺ CD25^high T cells were sorted from RORc^WT/WT or RORc^KO/KO mice and co-transferred together with naïve T cells in a ratio of 1:4. Mice were followed over 7 weeks. (A) Body weight change over the course of the experiment. (B-C) Histopathology in the colon tissue (B) and total cell number in the colon (C) in the different experimental groups. Data are shown as mean ± SD and represent one experiment with 5 mice per group.

from the colon of the individual mice and examined the cytokine production as well as the expression level of RORγt and Foxp3 in CD90.1⁺ T cells in the different organs (Figure 3.18 C, D). We detected an increased level of IL-17 and IFN-γ producing CD90.1⁺ Teff cells in the colon of mice co-transferred with either RORc^WT/WT or RORc^KO/KO Tregs; however, no differences could be observed when the two groups were compared. In correlation with these results we observed an increased frequency of CD90.1⁺RORγt⁺ Teff cells in the colon of mice co-transferred with either RORc^WT/WT or RORc^KO/KO Tregs. Again, no difference could be identified between the two groups. A modestly increased frequency of CD90.1⁺ Foxp3⁺ T

A

B C

cells could be detected in the group of mice co-transferred with RORc^WT/WT Tregs in comparison to the other groups. However, this difference was not significant (Figure 3.18 D).

spleen pLN mLN colon to induce colitis. Additionally, CD4⁺ CD25^high (CD90.2⁺) T cells were sorted from RORc^WT/WT or RORc^KO/KO mice and were co-transferred together with naïve T cells in a ratio of 1:4. Mice were followed over 7 weeks. (A-B) Spleen, pLN, mLN and colon were harvested, the lymphocytes were isolated, stained for CD90.2 and analyzed by flow cytometry. Frequency (A) and total number (B) of CD90.2⁺ T cells in the analyzed organs. (C-D) CD90.2^- Teff cells, isolated from the lamina propria of the colon, were stimulated, stained and analyzed for their expression levels of IL-17 and IFN-γ (C) or RORγt and Foxp3 (D) by flow cytometry. Data represent one experiment with 5 mice per group.

Based on these findings, we could not demonstrate that the increased suppressive capacity observed for RORγt⁺ Foxp3⁺ T cells in transfer colitis is RORγt dependent. However, the frequency of RORγt⁺ Foxp3⁺ T cells in the total Treg pool might be too small to cause a significant difference in disease outcome. A similar approach, co-transferring RORγt^- Foxp3⁺ T cells or total (RORγt^- andRORγt⁺) Foxp3⁺ T cells together with naïve T cells into RAG2 KO mice also revealed no significant differences of the two groups in terms of disease suppression (data not shown). Further studies, e.g. using Foxp3 specific RORγt deficient mice, are needed to address this issue in greater detail.

A B

% of CD90.2- IL-17+T cells TregsKO/KO+ RORc TregsWT/WT+ RORc

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4 Discussion

Numerous publications have described the functional plasticity of the different CD4⁺ T cell populations and their relevance for health and disease. The population of RORγt⁺ Foxp3⁺ T cells was identified and first described in 2008 by two publications^89,129. The highest frequency of these cells was found in the small intestine and produces IL-17 and IL-10. IL-17 producing Foxp3⁺ Tregs found in the CCR6⁺ memory T cell population in patients suffering from ulcerative colitis and colon carcinoma were shown to suppress T cell activation and to stimulate the production of inflammatory cytokines in vitro¹²³. However, the specific phenotype and functional (inflammatory or regulatory) capacity of these “inflammatory Tregs”

in vivo remained unclear. Moreover, their origin as well as their stability has not been examined so far. Our data showed that RORγt⁺ Foxp3⁺ T cells could originate from Tregs and represent a stable T cell population which shows characteristics of Tregs and Th17 cells.

They possess a marker gene expression of recently described effector Tregs¹⁹⁹ and display a significantly increased suppressive capacity in a model of experimental colitis in comparison to Foxp3⁺ T cells.

Previous studies have demonstrated that this specific T cell population could be induced by microbiota or cytokine (IL-6) derived stimuli^119,132. RORγt⁺ Foxp3⁺ T cells expanded under inflammatory conditions and displayed comparable suppressive capacity in comparison to RORγt^- Foxp3⁺ T cells in vitro¹²⁹. The differentiation of both Th17 cells and Tregs depends on TGF-β, leading to a co-expression of RORγt and Foxp3 in developing T cells. However, Foxp3 inhibits the function of RORγt, resulting in Treg differentiation in the absence of further cytokine stimuli by IL-6, IL-21 and IL-23⁸⁹. Therefore it was questionable if the population of RORγt⁺ Foxp3⁺ T cells represents an intermediate state in the differentiation of naïve T cells to Th17 cells or Tregs, an adaptation of Tregs to the intestinal environment or a conversion of Tregs to Th17 cells (or vice versa). Moreover, based on the direct inhibition of RORγt function by Foxp3, the consequence of RORγtexpression remains to be defined.

The ex vivo isolation and the analysis of RORγt⁺ Foxp3⁺ T cells was previously based on the expression of the reporter signal for RORγt (using to RORc(gt)-Gfp^TG mice¹²⁹) and the surface expression of CD25. However, CD25 was also shown to be expressed by activated T cells¹⁹⁵. Therefore we aimed at specifically isolating RORγt⁺ Foxp3⁺ T cells based on the expression of the transcription factors by establishing the Foxp3^RFPRORγt^GFP reporter mouse.

The combination of the RORc(gt)-Gfp^TG and Foxp3-IRES-RFP (FIR)¹⁹¹ reporter mouse strains enables the simultaneous detection of RORγt-GFP and Foxp3-mRFP due to non-overlapping fluorescence spectra. Monomeric red fluorescent protein (mRFP) is a derivative of Discosoma red fluorescent protein (DsRed). Although it possesses a lower extinction coefficient (44,000 M^-1*cm^-1) and quantum yield (0.25), it matures > 10 times faster than

DsRed, which forms a tetramer structure. Furthermore, it shows minimal overlap with the excitation spectra of GFP due to a shift of excitation and emission peaks of ~25 nanometer (nm) in comparison to DsRed²³⁶. As expected, our first analysis of the Fopx3^RFPRORγt^GFP reporter mouse did not show overlapping excitation or emission spectra of GFP and mRFP.

Therefore, we generated an efficient tool to isolate and characterize the population of RORγt⁺ Foxp3⁺ T cells in more detail.